Each year in the United States alone there are approximately 50,000 new.cases of Parkinson's disease. Parkinson's disease (PD) results from the malfunction or death of brain cells responsible for the production of the neurotransmitter dopamine. Dopamine deficiency results in tremor and degraded motor control. The etiology of Parkinson's is complex, and in most cases probably involves a combination of environmental and genetic factors. The advent of powerful genetic techniques has allowed the rapid identification of genes potentially associated with the development of PD. However, the subsequent biochemical characterization of the proteins encoded by these genes has been a bottleneck in understanding their roles in disease. The major goal of this proposal is to develop a new approach that will facilitate the characterization of PD associated proteins. The disruption of gene expression is an extremely powerful approach for defining the physiological roles of gene products. Recently, a technique known as RNA interference has allowed suppression of gene expression in a variety of experimental systems. However, it is often not possible to entirely eliminate the expression of a protein using this approach. Alternatively, gene sequences can be disrupted directly using a process known as homologous recombination. This approach offers complete and permanent disruption of targeted genes but has worked well only in a limited number of cell lines. We have modified the approach to potentially allow its use in many additional cell lines, including cell lines useful in PD research. As proof of principle, we will disrupt the gene encoding dardarin in a neuronal cell line. Although the function of dardarin is currently unknown, mutations in the gene (LRRK2) encoding the protein are associated with approximately 5% of all familial cases of Parkinson's disease. Using the dardarin deficient cell line, we will begin to examine the role of this novel protein in the physiology of neuronal cells. The development of a homologous recombination approach amenable to neuronal cell lines will facilitate the characterization of other PD-associated proteins. Characterization of the physiological roles of known PD associated proteins will lead to the identification of the biochemical pathways susceptible to damage in dopamine-producing neurons. This knowledge will aid in the identification of the environmental triggers and additional genetic factors that participate in the development of Parkinson's disease.